POS1072 INSULIN INHIBITS ACTIVITY OF CD4+ T CELLS IN RHEUMATOID ARTHRITIS
Authors: M. Erlandsson, M. Tuameh, L. Nilsson, E. Malmhäll-Bah, S. Silfverswärd, T. Zverkova Sandstrom, K. M. Andersson, M. Dehlin, R. Pullerits, M. I. Bokarewa
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Rheumatoid arthritis (RA) and diabetes mellitus (DM) have common genetic risk factors, while molecular mechanisms behind this commonality have not been studied. <h3>Objectives</h3> To study non-metabolic effects of insulin in CD4+ T cells of RA patients. <h3>Methods</h3> Incidental DM was studied in a consecutive cohort of 330 (256 female, 74 male) RA patients and 1011 (798 female, 213 male) gout patients matched to RA by age and gender (1 to 3) retrieved from the Health Care Register of Western Sweden during 10 years of follow-up. Insulin was measured at baseline and analyzed in relation to clinical and serological disease parameters. Transcriptomics by RNAseq was done on CD4<sup>+</sup>T cells of 57 RA patients. Differentially expressed genes (DEG, nominal p&lt;0.05) in patients with hyperinsulinemia (n=11, median 344 pmol/L) were identified using DESEq2 (R studio, Bioconductor) and analyzed for enrichment of biological processes and transcription factor (TF) families using GSEA (Broad Institute). To study direct effect of insulin on CD4<sup>+</sup>T cell function, <i>in vitro</i> culture experiments were performed. Analysis of CD4<sup>+</sup>Tcell phenotype for senescence, exhaustion and apoptosis was done by flow cytometry, transcriptomics and cytokine measurement. <h3>Results</h3> In total, 102 new cases of DM were registered, significantly less often in RA patients compared to gout patients (p=3.2e-4, OR=0.34 [0.18-0.64]). The new DM were more prevalent in men (p=0.012) with no difference between RA and gout patients (9.8 vs 7.5%, OR=1.34 [0.46-3.52] p=0.55). In females, RA patients developed significantly fewer cases of DM than gout patients (2.1 vs 9.4%, OR=0.21 [0.07; 0.47], p=3.2e-5). In RA patients, the insulin levels correlated positively to inflammation measured by serum levels of IL6, VEGF and IL8 followed by IFNγ total, 102 new cases of DM were registered, significantly less often in RA patients compared to gout patients (p=3.2e-4, OR=0.34 [0.18-0.64]). The new DM were more prevalent in men (p=0.012) with no difference between RA and gout patients (9.ol/L). Transcription profile of CD4<sup>+</sup>T cells revealed 688 (131 upregulated, 557 downregulated) protein-coding DEG in patients with hyperinsulinemia. The down-regulated DEG showed enrichment for T cell activation (GO:0042110, FDR 2.6e-26) and inflammatory response (GO:0006954, FDR 2.9e-21). Analysis for TF families showed that the down-regulated DEG were controlled by TFs HMGB1, NFAT and NFκB (all FDR&lt;e-6), which were significantly enriched for Th1 T cell markers including TBX21, EOMES, CXCR3 and IL2. The upregulated DEG were controlled by TFs MORC2, which regulates response to DNA damage, and ZBTB7B essential for differentiation and commitment of CD4<sup>+</sup>T cells. DEG included GART gene controlling the folate biosynthesis and the transcription regulating proteins MRPL3 and DAP3. Patients with hyperinsulinemia had significantly higher serum levels of IL6, IL8 and VEGF (all p&lt;0.05), suggesting an inflammatory environment. However, they were not different in DAS28, CRP, WBC and platelet counts. Stimulation of CD4<sup>+</sup>T cell cultures with insulin (n=12) resulted in a significant suppression of IFNγ cell cultures with insulin (n=12) resulted in a significant suppression of IFNscription regulating proteins MRPL3 and DAP3. Patients with hyperinsulinemia had significantly higher serum levels of IL6, IL8 and VEGF (all p&lt;0.05), suggesting an inflammatoryrichment for DNA (7AAD<sup>+</sup> cells) in flow cytometry, which does not support induction of senescence by insulin in CD4<sup>+</sup> cells. In contrast, CD4<sup>+</sup> cells of hyperinsulinemic RA patients expressed lower levels of CCL chemokines, IL2, and TNF, which suggests that insulin induced exhaustion of CD4<sup>+</sup>T cells. <h3>Conclusion</h3> This study shows that hyperinsulinemia has non-metabolic functions under conditions of chronic inflammation. It could indicate residual RA activity. Insulin controls overactivation of adaptive immunity and causes exhaustion of pro-inflammatory Th1 cells in RA patients. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.